The SPIM technique (single plane illumination microscopy), in which the sample is illuminated in layers, allows image data to be sensed more quickly and with less sample impact than, for example, with spot-type scanning of a sample. One known area of application of SPIM technology is the field of fluorescence microscopy, fluorophores in the sample being excited with laser light. In SPIM technology, excitation occurs only in an illumination light sheet (also called a “light strip”). Damage to the sample resulting from illumination light in other planes is thereby avoided.
An optical apparatus operating according to the SPIM method is described in DE 102 57 423 A1. With this microscope a sample is illuminated with a thin light strip while observation occurs perpendicularly to the plane of the illuminating light strip. Illumination and detection occur here via two separate optical beam paths each having separate optics, in particular having two separate objectives perpendicular to one another. The light strip is generated by an illumination objective and by a cylindrical optic placed in front of it. For image acquisition, the sample is moved through the light strip, which is stationary with reference to the detector, in order to acquire fluorescent light and/or scattered light in layers using a planar detector. The layer image data thereby obtained can then be assembled into a data set corresponding to a three-dimensional image of the sample. This document does not disclose manipulation of a sample.
DE 20 2011 110 077 U1 discloses an arrangement for illuminating a sample in the context of SPIM microscopy. The arrangement comprises a light source for generating a light bundle, means for generating a light strip from the light bundle, and at least one objective which comprises an optic that is embodied and intended to deliver detected light proceeding from the sample to a detector, directly or indirectly. The arrangement furthermore comprises a diverting device, located after the optic of the objective, for diverting the light strip.
The combination of SPIM technology and optical sample manipulation is very attractive in principle, since the very fast SPIM technology, with low sample impact, is very well suited for observing the effects of manipulations of, in particular, relatively large, living organisms.
It is known from DE 10 2007 047 464 A1 to make available an additional manipulation light source whose manipulation light is coupled via a lens arrangement and mirror arrangement into the illumination beam path via an illumination objective. Alternatively, the same document proposes additionally coupling manipulation light into the detection beam path by means of dichroic mirrors, and directing it through the detection objective onto the sample.
In an embodiment of this kind, in which manipulation light is delivered to the sample both via the illumination beam path and via the detection beam path, three beam splitters and two diaphragms must be laboriously inserted into the beam path. An advantage of this setup is that manipulation can take place from two directions. On the other hand, this results in a complex optical configuration and in light losses, at least on the illumination side.
The scientific publication “Selective plane illumination microscopy techniques in developmental biology,” Huisken et al., Development 136, 1963-1975 (2009) discloses an apparatus of this kind in which a photomanipulation laser whose light is focused via the detection objective onto the sample is additionally provided. A largely similar arrangement is known from “Three-dimensional laser microsurgery in light-sheet based microscopy (SPIM),” Engelbrecht et al., Optica Express 6420, Vol. 15, No. 10 (2007). The article by Yanik et al., “Technologies for Micromanipulating, Imaging, and Phenotyping Small Invertebrates and Vertebrates,” Annu. Rev. Biomed. Eng. 2011; 13: 185-217 also discloses a similar apparatus and method in which the samples are pumped through a capillary.